CA2456239C

CA2456239C - Intermediates for preparing neuraminidase inhibitor conjugates

Info

Publication number: CA2456239C
Application number: CA2456239A
Authority: CA
Inventors: Brian David Judkins; Simon John Fawcett Macdonald; Derek Anthony Demaine; Graham George Adam Inglis; Julie Nicole Hamblin
Original assignee: Biota Scientific Management Pty Ltd
Current assignee: Biota Scientific Management Pty Ltd
Priority date: 2001-09-07
Filing date: 2002-08-30
Publication date: 2010-11-30
Anticipated expiration: 2022-08-30
Also published as: EP1423385A1; EP1423385B1; US7230114B2; EP1423385A4; US20050032853A1; JP4463553B2; DK1423385T3; WO2003022841A1; PT1423385E; CA2456239A1; ATE399779T1; DE60227386D1; ES2307776T3; JP2005501921A; BR0212261A; MXPA04002141A

Abstract

Compounds of formula (I), methods for their preparation and their use in the manufacture of neuraminidase inhibitor conjugates. Wherein R represents as carboxylic acid protecting group; P1 and P2 can be the same or different and are selected from amine protecting groups; P3 represents a protecting group for 1, 2 diols; and LG represents a leaving group.

Description

INTERMEDIATES FOR PREPARING NEURAMINIDASE INHIBITOR CONJUGATES
The present invention relates to novel compounds, methods for their preparation and their use in the manufacture of neuraminidase inhibitor conjugates.
Dimeric compounds and their use as neuraminidase inhibitors have been disclosed in W000/55149. Polymeric compounds and their use as neuraminidase inhibitors have been disclosed in W098/21243. In W000/55149, it was shown that when two neuraminidase-binding compounds are suitably linked together through a region of the molecule that is not involved in binding to the active site, the resultant dimers have enhanced anti-viral activity. Eur. J. Med. Chem 34 (1999) 563-574 discloses the synthesis and influenza virus sialidase inhibitory action of an analogue series of 4-guanidino-Neu5Ac2en (zanamivir) modified in the glycerol side chain.

In W000/55149, compound 7 is described as a useful precursor to certain dimeric neuraminidase inhibitors.

H2N(CH2)6NH20 HO
HO
AcHN =
NH

Compound (7) We have found that in a first aspect the invention provides compounds of formula (I):

~_LG
O O
H
O
OR
P3\
O
AcHN =
NH
P2NHLNP1 (I) wherein R represents a carboxylic acid protecting group;

P1 and P2 can be the same or different and are selected from amine protecting groups;
P3 represents a protecting group for 1,2 diols; and LG represents a leaving group, for example, para-nitrophenol or a derivative thereof, halide, imidazole or N-hydroxysuccinimide.

Preferably R is C1_6 alkyl, diphenylmethane or an appropriate protecting group selected by one skilled in the art from common carboxylic acid protecting groups such as those listed in "Protective Groups in Organic Synthesis," TW Greene and PGM Wuts 1999 (3d edition), Wiley.
When used herein a C1_6 alkyl group can be straight, or branched, for example methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, or hexyl, preferably methyl or t-butyl. An alkyl group may also be cyclic, that is a C3-6 cycloalkyl group, for example cyclohexyl.

Common amine protecting groups are as those listed in "Protective Groups in Organic Synthesis,"
TW Greene and PGM Wuts 1999 (3d edition), Wiley, preferably a t-butoxycarbonyl (Boc) group.
Protecting groups for 1,2 diols are CO (a cyclic carbonate) or CHMe (a methyl acetal) or an appropriate protecting group selected by one skilled in the art from common 1,2 diol protecting groups such as those listed in "Protective Groups in Organic Synthesis," TW
Greene and PGM
Wuts 1999 (3d edition), Wiley. Preferably P3 represents CO or CHMe.

Other leaving groups will be known to the person skilled in the art for the preparation of carbamates.

Even more preferably R is methyl or diphenylmethane, P1 and P2 are Boc, P3 is CO and LG is para-nitrophenol.

Compounds of the present invention offer a significant advantage in the rapid preparation of large numbers of neuraminidase inhibitor conjugates, specifically those disclosed in WO 00/55149.
Compounds of the present invention provide a common intermediate from which a large number of neuraminidase inhibitor conjugates can be prepared using different "linking groups" many of which are commercially available. Using a common intermediate allows flexibility and the ability to produce large numbers of compound quickly.

Compounds of formula (I) may be useful in the preparation of compound libraries comprising at - 2a-least 2, e.g. 5 to 1000, compounds, preferably 10 to 100 compounds. Compound libraries maybe prepared by "split and mix" approach or by multiple parallel synthesis using either solution phase or solid phase chemistry, by process known in the art.

A second aspect of the invention is the use of compounds of formula (I) in the preparation of neuraminidase inhibitor conjugates, specifically those disclosed in WO
00/55149.

A third aspect of the invention is the process for the preparation of neuraminidase inhibitor conjugates, specifically those disclosed WO/00155149 comprising the use of compounds of formula (I).

A further aspect of the invention is neuraminidase inhibitor conjugates, specifically those disclosed in W000/55149, prepared using compounds of formula (I).

W000/55149 and W098/21243 teaches the generic formula of the neuraminidase inhibitor conjugates.

Compounds of formula (I) can be prepared by reaction of compounds of formula (III):
O
OR
3\ O
AcHN (III) NH
P=HN PI

wherein PI, P2, P3 and R are as described for compounds of formula (I), with compounds of formula (II):

La (n) wherein each LG is independently as described for compounds of formula (I), in a solvent, and a base.

Preferably the base used is a tertiary amine, for example dimethylaminopyridine (DMAP), 4-pyrrolidinopyri dine or 1,8-diazabicyclo[5.4.0]undec-7-ene, more preferably DMAP.
Preferably at least two equivalents of base to compound of formula (III) are used.

Preferably the solvent is pyridine or a pyridine type solvent.

Preferably the reaction should be carried out in the absence of water, for example by azetroping the starting materials, or drying in an oven prior to carrying out the reaction.
For example compounds of formula (II) may be symmetrical or unsymmetrical e.g.
p-nitrophenylchloroformate.

Compounds of formula (III) can be prepared by reaction of compounds of formula (IV);

H
HO O
OR
HO
AcHN =
NH
(IV) wherein P1, P2 and R are as described for compounds of formula (I), with carbonyldiimidazole (CDI) or phosgene or other phosgene equivalents.
Compounds of formula (IV) wherein R is diphenylmethane are known in the literature, J
Med Chem 1998, 41, 787-797.

Neuraminidase inhibitor conjugates of formula (V);

O Linker N O --f H O O

O

O
H O
0 0 MeO O
OMe 0 0 NHAc AcHN NH
NH (V) HN NH

may be prepared by reacting compounds of formula (I) with compounds of formula (VI);
H2N-_ Linker NH2 NO

-in solvent, for example pyridine, and in the presence of base, for example DMAP, followed if necessary by deprotection.

5 Methods of deprotecting the amine and ester groups will be well known to the person skilled in the art.

When used herein halide represents a fluoro, chloro, bromo or iodo group.

Compounds of formula (V) can be tested for neuraminidase activity by methods known in the art for example by plaque assays, Hayden et al. (Antimicrobial. Agents Chemother., 1980, 17, 865).

The invention will now be described in detail by way of reference to the following non-limiting examples.

Examples 1 and 2 disclose the preparation of compounds of formula (I). Example describes the preparation of a neuraminidase inhibitor conjugate of formula (V).

Abbreviations used herein are DPM -diphenylmethane SPE - solid phase extraction.
DMAP - 4-dimethylaminopyridine BOC - t-butoxycarbonyl EtOAc - ethyl acetate DCM - dichloromethane THF- tetrahydrofuran CDI- 1,1'-carbonyldiimidazole LC/MS liquid chromatography mass spectrometry.
Example 1.
Intermediate 1 Benzhydryl (2R,3R,4S)-3-(acetylamino)-4-(J [(tert-butoxycarbonyl)amino] [(tert-butoxycarbonyl)imino]methyl } amino)-2- (S)-hydroxy [(4R)-2-oxo-1,3-dioxolan-4-yl] methyl } -3,4-dihydro-2H-pyran-6-carboxylate OH OH OH

OH MeCN

HN = HN
0 HN,NHBOC CDI 0 0 HNY NHBOC
NBOC Int 1 NBOC
C35H46N4011 = 698 C,H44N4O12 = 724 Benzhydryl (2R,3R,4S)-3-(acetylamino)-4-Q (E)-[(tert-butoxycarbonyl)amino]
[(tert-butoxycarbonyl)imino]methyl } amino)-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylate (see J. Med. Chem. 1998, 41, 787-797) (12.38g;
17.7mmoles) was dissolved in dry acetonitrile (130ml) under nitrogen at room temperature.
The solution was stirred and 1,1'-carbonyldiimidazole (2.87g; 17.7mmoles) was added. After 16 hours LC/MS showed the presence of starting triol so further 1,1'-carbonyldiimidazole (total of 0.493g; 3mmoles) was added. After a few hours LC/MS showed no triol present.
The solvent was evaporated and the residue flash columned on silica, eluting with 1:1 ethyl acetate/40-60 petroleum ether. Fractions containing wanted product were evaporated then taken up in dichloromethane, dried with sodium sulphate, filtered and evaporated to give Intermediate 1 as an off white solid (11.05g; 86%).

LC/MS (Blue method) MH+ = 725, Tet = 4.09 minutes.
Example 1 Benzhydryl (2R,3R,4S)-3-(acetylamino)-4-({ (E)-[(tert-butoxycarbonyl)amino] [(tert-butoxycarbonyl)imino]methyl } amino)-2- { (S)- {
[(4-nitrophenoxy)carbonyl]oxy } [(4R)-2-oxo-1,3-dioxolan-4-yl]methyl } -3,4-dihydro-2H-pyran-6-carboxylate O

~~HN
HN~N~O
O
O"N O`

A solution of benzhydryl (2R,3R,4S)-3-(acetylamino)-4-({ [(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl } amino)-2- { (S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl }-3,4-dihydro-2H-pyran-6-carboxylate (Intermediate 1)(143mg, 0.197mmol) in dry pyridine (3m1) containing 4-dimethylaminopyridine (120mg, 0.982mmo1) was treated with 4-nitrophenylchloroformate (199mg, 0.987mmol) at 22 C. The mixture was stirred at 22 C. For 17h, then the pyridine removed in vacuo. The residue was purified by SPE
chromatography (5g cartridge) eluting with cyclohexane - ethyl acetate (4:1 - 2:1) to afford Example 1 as a pale yellow gum (99mg, 56%).

NMR 8(CDC13) 11.30 (lHbrd, NH), 8.62 (l H brd, NH), 8.23 (2H, AA' BB', aromatic CH's), 7.52 (2H, AA'BB', aromatic CH's), 7.43-7.30 (10Hm, aromatic CH's), 6.95(lHs, CH), 6.76 (111 brd, NH), 6.05 (lHd, =CH), 5.56 (lHdd, CH), 5.22 (lHdt, CH), 5.00 (lHdt, CH), 4.72 (lHdd, CH), 4.59 (lHdd, CH), 4.48 (lHq, CH), 4.25 (lHdd, CH), 1.92 (3Hs, CH3), 1.48 (9Hs, tert butyl), and 1.43 (9Hs, tert butyl).
LCIMS Rt = 4.19min. (MH = 890, MH" = 888) Example 2-OH OH OH OH

H O I C02H HCI/MeOH H O C02Me OH OH
HN
0-J\ NH2 Int 2 O-J\ NH2 .HCI
C,,H18N207 =290 C72H2ON201=304 N NBOC

NHBOC
THF/MeOH/Et3N
OH OH OH

H O C02Me ::N HN =

0 HNrNHBOC 0-_~\ HNYNHBOC
Into NBOC Int 3 NBOC
C24H36N4O12=572 C23H38N401,=546 Intermediate 2 Methyl (2R,3R,4S)-3-(acetylamino)-4-amino-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylate hydrochloride Acetyl chloride (75m1; 1.05mole) was added drop-wise with stirring to methanol (7500m1) at 0-5 C under nitrogen. The mixture was stirred at this temperature for a further 15 minutes then held at approximately 10 C as (2R,3R,4S)-3-(acetylamino)-4-amino-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylic acid trihydrate (see J. Med. Chem. 1998, 41, 787-797) (250g; 726mmoles) was added in portions. The mixture was stirred at approximately 60 C for 5 hours then cooled to approximately 20 C and stirred at this temperature overnight. The solvent was removed and the residue twice evaporated down again with methanol (2x500m1) to give a mixture of a foam and gum. This was re-dissolved in methanol (-1000m1), evaporated and the residue then triturated with DCM and re-evaporated. The trituration DCM
process was repeated. The residue was dried overnight in a vacuum-oven at approximately 30 C, crushed and then dried overnight again to give Intermediate 2 as a white powder (264.2g).

LC/MS (Orange Method) MH+ = 305, Tret = 0.54 minutes.
Intermediate 3 Methyl (2R,3R,4S)-3-(acetylamino)-4-Q [(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylate The amino ester hydrochloride Intermediate 2 (211.6g; 0.62mole) was added portion-wise to methanol (2100m1) stirring under nitrogen in a 10 litre reactor to give a pale brown solution. THE (2100m1) was added. Triethylamine (86.5m1; 0.62mole) was added drop-wise with stirring and then a solution of N,N'-bis-t-butyloxycarbonyl-l-guanylpyrazole (201.3g; 0.649mmole) in THE (2100m1) was added drop-wise, fairly quickly, maintaining the reaction temperature at approximately 22 C. The mixture was stirred under nitrogen at approximately 22 C for 45 hours then filtered to remove a small amount of solid and the filtrate evaporated to dryness. After standing overnight the gummy yellow residue was triturated with ethyl acetate (2500m1) by rotation on rotary evaporator to give a fine white solid which was filtered off. The filtrate was evaporated down and dried under high vacuum to give a foam (-333g). The foam was dissolved in 3% methanol/DCM
(-700m1) and purified on a 2.5kg Biotage column pre-conditioned in and eluted with 3%
methanol/DCM. The purest fractions were combined and evaporated then dried at approximately 30 C to give Intermediate 3 as a white solid (192.8g; 49.4%
yield corrected for the presence of pyrazole). NMR showed the presence of -54mole %
pyrazole (-13% by weight).

LC/MS (Orange Method) MH+ = 547, Tre1= 5.07 minutes.
Intermediate 4 methyl (2R,3R,4S)-3-(acetylamino)-4-({ [(tert-butoxycarbonyl)amino] [(tert-butoxycarbonyl)imino]methyl } amino)-2- { (S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl } -3,4-dihydro-2H-pyran-6-carboxylate Intermediate 3 (423.2g; ca 0.77mole) (contaminated with -13% pyrazole), was dissolved in dry acetonitrile (4750m1) and stirred under nitrogen in a 10 litre reactor.
CDI (135.6g;
0.84mole) was added portion-wise using circulator to control the slight exotherm and maintain the reaction temperature at approximately 22 C. The mixture was stirred at this temperature under nitrogen overnight. After 22 hours the solvent was removed and the residual yellow gum was dissolved in ethyl acetate (3500ml) and returned to the reactor.
The solution was washed in the reactor twice with dilute hydrochloric acid (2x1250m1;
1M), then once with water (1000ml), then once with brine (800m1). The solution was dried over magnesium sulphate, filtered, evaporated and dried in high vacuum to give a white foam (378g). The foam was dissolved in DCM (--1000ml) and the solution applied in two batches to a 2.5kg Biotage column preconditioned in and eluted with 1:1 hexane/ethyl acetate to give, after evaporation and drying, Intermediate 4 as a white solid (total 292.1g; -76% based on corrected amount of starting material).

LCIMS (Orange Method) MH+ = 573, Tret = 5.85 minutes.

Example 2- Methyl (2R,3R,4S)-3-(acetylamino)-4-({(E)-[(tert-butoxycarbonyl)amino] [(tert-butoxycarbonyl)imino]methyl }amino)-2- { (S)-{
[(4-nitrophenoxy)carbonyl]oxy } [(4R)-2-oxo-1,3-dioxolan-4-yl]methyl } -3,4-dihydro-2H-pyran-6-carboxylate O
O---~

H

O
O=-O HN
HN~Ny0 O 111, 0 Y N 0`

A solution of methyl (2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl } amino)-2- { (S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl } -3,4-dihydro-2H-pyran-6-carboxylate (113mg, 0.197mmol) in dry pyridine (3m1) containing 4-dimethylaminopyridine (120mg, 0.982mmo1) was treated with 4-nitrophenylchloroformate (199mg, 0.987mmo1) at 22 C. The mixture was stirred at 22 C. For 17h, then the pyridine removed in vacuo. The residue was purified by SPE chromatography (5g cartridge) eluting with cyclohexane - ethyl acetate (4:1 - 2:1) to afford Example 2 as a pale yellow gum (96mg, 66%).
NMR S(CDC13) 11.3 (1Hs, NH), 8.58 (1H brd, NH), 8.26 (2H, AA'BB', aromatic CH's), 7.56 (2H, AA'BB', aromatic CH's), 6.82 (1H brd, NH), 5.93 (lHd, =CH), 5.54 (1Hdd, CH), 5.20 (1 Hdt, CH), 5.10 (1 Hdt, CH), 4.78 (2Hm, 2xCH), 4.44 (l H brq, CH), 4.28 (1 Hdd, CH), 3.82 (3Hs CH3), 1.91 (3Hs, CH3), and 1.48 (18Hs, 2x tert butyl).
LCMS R, = 3.87min. (MH+ = 738, MHf = 736) Example 3 OH OyO & NO2 H O COZDPM
4-NO2 PhOCOCI 0 o o H O C02DPM
II
0 HN HN NHBOC pyridine/DMAP 10 Oo 0 u u HN
II
NBOC
C36H44N4012 = 724 C43H47N5016 = 889 H2N__~O-'-I-O---_-O-__-I-_\NH2 O)LN~~\O~/O~\O~\N)LO
H
OH C02DPM H O ( COZDPM
OYO OYO
HN HN
O 0 HNyNHBOC 0 O J HNYNHBOC
NBOC J~ NBOC
C84H103N10 29 = 1720 The benzhydryl (2R,3R,4S)-3-(acetylamino)-4-Q [(tert-butoxycarbonyl)amino]
[(tert-butoxycarbonyl)imino]methyl } amino)-2- { (S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate (0.4g;0.55mmole) was azeotroped times from dry toluene and the dried solid was dissolved in molecular sieve-dried pyridine (1.6m1). The solution was treated with 4-dimethylaminopyridine (0.17g;1.4mmoles). To this was added 4-nitrophenylchloroformate (0.12g;0.6mmole) under nitrogen. A slight exotherm occurred, the temperature rising from 24 C
to 27 C.
The mixture was stirred at room temperature for 3 hours after which time LC/MS
showed the absence of starting material and the presence of the nitrophenylcarbonate (Example 1) MH+ = 890.
To this mixture was added 4,7, 1 0-trioxa- 1, 13-tridecanediamine (60.7mg;
0.276mmole) in dry pyridine (1 ml). The resulting mixture was stirred at room temperature for 3 hours after which time LCMS showed the absence of the nitro compound 2 and the presence of product 3 at (M + 2H+)/2 = 861. Volatiles were removed in vacuo at 40 C and the resulting orange oil was applied to a lOg Si SPE cartridge eluted with DCM(5x), ether(5x) and EtOAc(5x). The product eluted in the EtOAc fractions as a white solid (0.2g; 22%).

The product may be deprotected using standard techniques.

N.B. The 4-nitrophenylchloroformate should be white with no trace of yellow colour.
LC/MS Details - Blue Method Micromass Platform II mass spectrometer operating in positive ion electrospray mode, mass range 100-1000 amu.
Column : 3.3cm x 4.6mm ID, 3 m ABZ+PLUS
Flow Rate : 3m1/min Injection Volume : 5p.1 Solvent A : 95% acetonitrile + 0.05% formic acid Solvent B : 0.1 % formic acid + 1OmMolar ammonium acetate Gradient : 0% A/0.7min, 0-100% A/3.5min, 100% A/l.lmin, 100-0% A/0.2min LC/MS Details - Orange Method Instrument: Micromass Platform II
Ionisation Mode: Electrospray +ve Range: 100-1000amu Column: 50mm x 2.1mm Phenomenex Luna C18, 5um.
Flow:1.0ml/min Inj Vol: 5u1 Diode Array Detector: 220-300nm Mobile Phase: A - Water + 0.05% v/v TFA.
B - Acetonitrile + 0.05% v/v TFA
Gradient: Time %A %B

8.1 100 0 It is to be understood that the present invention covers all combinations of particular and preferred subgroups described hereinabove.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word `comprise', and variations such as `comprises' and `comprising', will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein.
They may take the form of composition, process, or use claims and may include by way of example and without limitation the following claims.

Claims

1. A compound of formula (I):

wherein R represents a carboxylic acid protecting group;
P1 and P2 can be the same or different and are selected from amine protecting groups;
P3 represents a protecting group for 1,2 diols; and LG represents a leaving group.

2. A compound according to claim 1, wherein R is C1-6 alkyl, C3-6 cyclic alkyl or diphenylmethane.

3. A compound according to claim 1 or claim 2, wherein Pi and/or P2 is/are a t-butoxy carbonyl (Boc) group.

4. A compound according to any one of claims 1 to 3, wherein P3 is CO or CHMe.

5. A compound according to any one of claims 1 to 4, wherein LG is para-nitrophenol, halide, imidazole or N-hydroxysuccinimide.

6. A compound according to any one of claims 1 to 5, wherein R is methyl or diphenylmethane, P1 and P2 are Boc, P3 is CO and LG is para-nitrophenol.

7. A process for the preparation of a compound of formula (I) as defined in any one of claims 1 to 6, which comprises reacting a compound of formula (III):

wherein P1, P2, P3 and R are as defined in any one of claims 1 to 6, with a compound of formula (II):

wherein each LG is as defined in any one of claims 1 to 6, in a solvent, and a base.

8. A process as claimed in claim 7, wherein the reaction is carried out in the absence of water.

9. A process according to claim 7 or claim 8, wherein the solvent is pyridine.

10. A process according to any one of claims 7 to 9, wherein the base is a tertiary amine.

11. A process according to claim 10, wherein the tertiary amine is dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine or 1,8-diazabicyclo[5.4.0]undec-7-ene.

12. Use of a compound of formula (I) as defined in any one of claims 1 to 6, in the preparation of a neuraminidase inhibitor conjugate of formula (V):

13. A process for the preparation of a compound of formula (V) as defined in claim 12, which comprises reacting the compound of formula (I) as defined in any one of claims 1 to 6, wherein in the compound of formula (I), R is methyl and P3 is C=O, with a compound of formula (VI):

in a solvent and in the presence of base followed by deprotection.